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Rapid screening method for cocaine and benzoylecgonine in saliva samples
Analytica Chimica Acta 372 (1998) 349±355
Rapid screening method for cocaine and benzoylecgonine in saliva samples A.D. Campigliaa, T. Vo-Dinhb,* a
Department of Chemistry, North Dakota State University, Fargo, ND 58105-5516, USA Life Sciences Division, Advanced Monitoring Development Group, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101, USA
b
Received 12 January 1998; received in revised form 13 April 1998; accepted 28 April 1998
Abstract We are presenting a new method for cocaine and benzoylecgonine analysis in saliva samples based on room temperature phosphorimetry. The determination of the drugs is performed on ®lter paper, which is a suitable substrate for most saliva collection procedures. Extraction from the solid substrate is not necessary, since both compounds are detected on the paper substrate. Complete analysis can be performed with only a few microliters of saliva, which is an attractive feature in cases of limited sample availability. The limits of detection are estimated at the sub-ng level, which shows the feasibility of detecting cocaine and benzoylecgonine at the cut-off levels stipulated by the National Institute on Drug Abuse (300 ng/ml). When compared to the existing methodology, the phosphorimetric method presents several advantages which include simplicity, short analysis time, low cost, and the viability of interfacing it with portable instrumentation for ®eld screening applications. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Phosphorescence; Cocaine; Benzoylecgonine; Drug diagnostics; Saliva; Drug screening
1. Introduction For the past two decades, there has been an increasing interest in the use of saliva as a matrix of analysis for therapeutic drug monitoring in a variety of clinical situations [1]. In the speci®c case of drugs of abuse monitoring, the advantages of saliva over traditional ¯uids such as urine and blood include the elimination of privacy, and to a large degree, adulteration over sample collection. The collection of saliva is a noninvasive procedure which does not require special *Corresponding author. Tel.: 00 1 615 574 6249; fax: 00 1 615 576 7651; e-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00317-1
facilities and close supervision of private functions of the subject. Although the possibility of tampering with a sample by the donor can never be completely excluded, it is unlikely that any of the commonly used methods of adulteration of urine samples can be easily applied to saliva [2±4]. Particular interest, therefore, has been expressed by law enforcement agencies for illicit drug monitoring operations and for road-side testing of potentially intoxicated drivers [2,5,6]. Recent investigations have proved various degrees of correlation among the concentration levels of cocaine and benzoylecgonine in saliva with those encountered in blood and urine samples after oral and intravenous administration [7±10]. Although
350
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355
further studies are needed to completely understand the role of saliva as a diagnostic ¯uid for drugs of abuse, [4] the positive identi®cation of cocaine and benzoylecgonine at the ng/ml level have demonstrated the potential of saliva as a non-invasive matrix of analysis. Several methods have been reported for the identi®cation and quantitation of cocaine and its metabolites in saliva. These include immunoassay, [10±12] gas chromatography, [7] and gas chromatography±mass spectrometry (GC±MS) [9,10] Currently, immunoassay is the method most frequently employed for the identi®cation of cocaine and its metabolites in biological ¯uids. Although it is a useful approach to estimate cocaine concentrations in saliva samples, it cannot be considered as a reliable quantitative assay [11]. In addition, immunoassay procedures are elaborated and time-consuming, which are important limitations for routine, in situ analysis in ®eld monitoring operations. The increasing credibility of saliva as a non-invasive matrix of analysis, associated to the need for reliable techniques in clinical and forensic science, encouraged us to develop a new screening method for cocaine and benzoylecgonine in saliva. The new method is based on solid-surface room-temperature phosphorimetry (SS-RTP), an established analytical technique which provides selective, sensitive, and simple detection of a wide variety of organic compounds [13,14]. When compared to classical methods such as immunoassay and GC-MS, the new technology offers several advantages which include simplicity and short analysis time, low cost, and the possibility of interfacing it with portable instrumentation for ®eld screening applications [15,16]. 2. Experimental 2.1. Chemicals Whatman no. 1 chromatography paper and distilled, deionized water were used throughout this work. All the chemicals were analytical-reagent grade and used without further puri®cation. Cocaine hydrochloride and benzoylecgonine standards were purchased from Sigma (USA) in methanol at 1.0 mg/ml. Benzoic acid was acquired from Aldrich and methanol from Baker,
(USA). Sodium hydroxide was obtained from EM Science (USA). Sodium dodecyl sulfate (SDS; specially puri®ed for biochemical work), lead(II) acetate, thallium(I) nitrate, cadmium(II) chloride, and sodium iodide were from Sigma (USA). 2.2. Working solutions Cocaine, benzoylecgonine, and benzoic acid solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). 2% SDS, 1 M NaI, 0.5 M Pb(CH3CH2O2), 0.1 M TlNO3, 0.5 M AgNO3, and 1.5 M CdCl2 were prepared in water. Note: use extreme caution when handling thallium salts, which are known to be extremely toxic. Saliva samples were collected from four non-user volunteers using the spitting method [1]. A portion of each sample was either spiked with cocaine or benzoylecgonine. NaOH was added to the spiked and non-spiked samples to obtain ®nal solutions with 1 M NaOH concentration. In all cases, the solvent was methanol/water 50:50 (v/v), the volume of saliva was 1 ml, and the volume of analyte solution (3.94 10ÿ4 M) was 0.1 ml. 2.3. Background reduction treatment The treatment for paper background reduction consisted in a combination of water extraction and uv irradiation [17]. Paper strips were water extracted in a soxhlet apparatus for 8 h, and exposed to uv irradiation in a photochemical reactor for the same period of time. Once treated, the solid supports were then stored in a desiccator for use in all future analyzes. 2.4. Photochemical reactor The UV irradiation of paper substrates was performed in a Rayonet photochemical reactor (Southern NE Ultraviolet, Middletown, CT, USA) using ®ve lamps with maximum wavelength of emission (em) at 254 nm and seven with em at 300 nm. 2.5. Spectrofluorimeter The phosphorescence spectra and lifetimes were determined with a Perkin-Elmer LS-50B luminescence spectrometer. Excitation and emission slit
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355
widths were adjusted to provide 15 and 20 nm spectral resolution, respectively. In the acquisition of phosphorescence intensities, the gate time and the delay time were set at 9 and 0.3 m s, respectively. 2.6. Sample procedure Detailed procedures for the RTP assay have been discussed previously [13,14]. Although only the salient features are presented here, RTP assays usually involve simple and rapid experimental steps. All solutions were spotted on solid substrates using 2 ml micropipettes (Rainin, Model P2). In the cases were phosphorescence enhancers were used, the heavy atom and/or surfactant solutions were spotted prior to analyte deposition. In all cases, a volume of 1 ml was employed. The solid substrates were dried under an infrared lamp for 2 min. After drying, the samples were placed into a desiccator containing CaSO4 chips. All RTP measurements were performed under a ¯ow of dry nitrogen to avoid possible quenching effects from oxygen and moisture. A silica gel bed was used to remove water vapor traces from the drying ¯ow. 3. Results 3.1. Enhancement of phosphorescence emission on chromatography paper The delivery of methanolic solutions of cocaine hydrochloride and benzoylecgonine on paper substrates resulted in no RTP signal. Phosphorescence emission was induced in basic medium [18]. The analyte solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). Both compounds showed RTP emission with maximum excitation and emission wavelengths at 253 and 410 nm, respectively. Although the analyte signal intensities were relatively low (5.9410ÿ4 M analyte solutions gave signal-tobackground ratios ± IAB/IB ± approximately equal to 2), we were able to differentiate their spectral characteristics from the background signal of the paper substrate. Knowing that the basic hydrolysis of cocaine and benzoylecgonine leads to the formation of benzoic acid and ecgonine, [19] and considering that phos-
351
phorescence emission at room temperature is observed from saturated organic molecules with a certain degree of conjugation, we investigated the RTP characteristics of benzoic acid in 1 M NaOH (methanol/ water 50:50 v/v). As expected, its phosphorescence spectrum was equivalent to the one observed for cocaine hydrochloride and benzoylecgonine in basic medium. Although these two compounds have the benzoate chromophore in their molecular structure, their interaction with the solid substrate might not provide the analyte rigidity required for strong phosphorescence emission at room temperature. At 77 K, however, the radiationless deactivation of cocaine's triplet state is minimized by the frozen matrix, and the phosphorescence emission from the benzoate group is readily observed [20±23]. 3.2. External heavy-atom effect Five inorganic salts (1 M NaI, 0.5 M Pb(CH3CH2O2)2, 0.1 M TlNO3, 0.5 M AgNO3, and 1.5 M CdCl2) were tested as phosphorescence enhancers [13,14]. Table 1 shows the results obtained for cocaine hydrochloride in basic medium. Cd(II) and Ag(I) ions did not induce RTP emission from the studied compounds (IAB/IB1). Tl (I) promoted RTP enhancement (IAB/IB>1), but its enhancing effect was lower than the one observed with iodide ions. As expected, similar results were obtained for benzoylecgonine. All further studies, therefore, were Table 1 Heavy-atom effect on the RTP characteristics of cocaine hydrochloride in basic mediuma Inorganic saltb
excc (nm)
emd (nm)
IAB/IBe
1 M NaI 1.5 M CdCl2 0.1 M TlNO3 0.5 M AgNO3
253 Ð 230 Ð
410 Ð 400 Ð
7.80.9 1.00.1 3.80.8 1.00.2
a
2.9710ÿ4 M solution of cocaine hydrochloride in 1 M NaOH (methanol/water 50:50 v/v) was employed. One ml sample volume was spotted on low background chromatography paper. b All heavy-atom solutions were prepared in methanol/water (30:70 v/v). One ml heavy-atom solution was employed. c Maximum excitation wavelength. d Maximum emission wavelength. e IABanalyte signal; IBbackground signal. Six measurements of analyte signals and their respective blanks were performed to obtain the analyte-to-background signal ratios.
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performed employing 1 M NaI as a phosphorescence enhancer. 3.3. Surfactant effect As a tentative means of improving iodide ef®ciency, we tested SDS in the matrix of the phosphor [24]. A 2% SDS solution was spotted on the chromatography paper prior to 1 M NaI and analyte deposition. A signi®cant increase in the IAB/IB ratio was observed in the presence of surfactant. For a 2.9710ÿ4 M cocaine hydrochloride solution (prepared in 1 M NaOH; methanol/ water 50:50 v/v), an IAB/IB 15.71.6 was obtained (NABNB9). This ratio is approximately two times higher than the one obtained in the absence of surfactant (see Table 1), showing that SDS increases the phosphorescence enhancing ef®ciency of iodide ions. 3.4. Analytical figures of merit Table 2 shows the analytical ®gures of merit of cocaine hydrochloride and benzoylecgonine under experimental conditions for maximum phosphorescence emission. Each phosphorescence intensity plotted on the calibration graph was the average of six data points. The linear dynamic ranges (LDR) extended over three orders of magnitude. The slopes Table 2 Analytical figures of merit of cocaine hydrochloride and benzoylecgonine obtained under experimental conditions for maximum phosphorescence emissiona Compoundb
LDRc (ng)
Slope log±log
Cocaine hydrochloride 0.16±50 1.05 Benzoylecgonine 0.22±50 0.94 a
Correlation ALODd coefficient (ng) 0.9944 0.9987
0.16 0.22
Maximum phosphorescence emission was obtained on solid substrates successively spotted with 1 ml of 1 M NaI and 1 ml of a 2% aqueous solution of sodium dodecyl sulfate. The measurement wavelengths were 253 and 410 nm. b The analyte solutions were prepared in 1 M NaOH (methanol/ water 50:50 v/v). c LDRlinear dynamic range. It was estimated by dividing the upper linear concentration by the limit of detection. d ALODabsolute limit of detection. Sample volume: 1 ml. The LOD values were estimated from the equation CLksb/m, where the standard deviation (sb) from 16 blank determinations, the slope of the calibration curve (m) and k 3 were employed.
Table 3 Phosphorescence lifetimes of cocaine hydrochloride, benzoylecgonine, and benzoic acid on chromatography paper treated for background reductiona Compoundb
exc/em (nm)
p; Short componentc (m s)
p; Long componentd (m s)
Cocaine hydrochloride Benzoylecgonine Benzoic acid
253/410 253/410 253/410
0.600.05 0.620.01 0.680.05
2.260.20 2.140.48 2.100.10
a Solid substrates spotted with 1 ml of 1 M NaI and 1 ml of 2% sodium dodecyl sulfate were employed. b All solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). c Delay times were from 0.1 to 1.1 m s. Gate time was 0.1 m s. d Delay times were from 2 to 9 m s. Gate time was 0.1 m s.
of the log±log plots were close to one, which showed a linear relationship between phosphorescence intensity and analyte concentration. The correlation coef®cients of the calibration curves were close in unity, indicating a satisfactory precision of measurement. For both compounds, the relative standard deviations were better at medium and higher concentrations, varying from 4.8% (for a 10 mg/ml cocaine solution) to 8.7% (for a 50 mg/ml benzoylecgonine solution). The phosphorescence lifetimes of cocaine hydrochloride, benzoylecgonine, and sodium benzoate in basic medium are shown in Table 3. The exponential decays were complex and included short and long decaying components. Their values were calculated by the stripping method suggested by Demas [25]. The least-squares analysis of the long decay components yield correlation coef®cients close in unity (higher than 0.99). The lifetime values in Table 3 are based on six determinations (N6) of the analyte decay and the blank decay. For a con®dence interval of 95% (P0.05), the statistical equivalence [26] among the lifetimes of the three compounds support our initial assumption that sodium benzoate is the phosphorescent species in basic medium. 3.5. Determination of cocaine hydrochloride and benzoylecgonine in saliva Saliva samples were collected from four non-user volunteers. Two tobacco smoker volunteers were included in the test group. The RTP measurements were performed with 1 ml of saliva deposited on
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355
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Fig. 1. (A) Excitation and (B) emission spectra of saliva samples (a) spiked and (b) non-spiked with cocaine hydrochloride. The amount of cocaine hydrochloride deposited on the paper substrate is approximately 11 ng.
chromatography paper previously treated with 1 M NaI and 2% SDS. The phosphorescence spectra of the spiked and non-spiked samples were compared for the four individuals. Fig. 1 shows the RTP spectra of cocaine in saliva. The presence of cocaine in the spiked sample was clearly detected by the phosphorescence bands with maximum excitation and emission wavelengths at 258/413 nm, respectively (see Fig. 1, curve A). The phosphorescence spectrum of saliva showed a broad excitation band with plateau extended from 240 to 280 nm, and a maximum of emission at
440 nm (Fig. 1, curve B). The contribution of saliva to the background signal of the solid substrate was approximately 15%. This contribution did not deteriorate cocaine's limit of detection in saliva, which was estimated at the ng level. Equivalent results were obtained for benzoylecgonine (P0.05; N6) [26]. No signi®cant differences were observed in the spectral characteristics of the saliva samples collected from the four volunteers. Table 4 summaries the lifetimes of cocaine phosphorescence emission in saliva samples. The values in
Table 4 Phosphorescence lifetime measurements of cocaine in saliva samplesa Volunteer
1 2 3 Average RSD (%)b a b
Spiked samples
Non spiked samples
Short component (ms)
Long component (ms)
Short component (ms)
Long component (ms)
0.83 1.00 0.90 0.910.08 9.3
4.17 4.22 4.25 4.210.04 1.0
2.38 2.81 3.06 2.750.34 12.4
5.72 5.62 5.70 5.680.05 0.9
For experimental conditions and instrumental parameters see Table 2. RSDrelative standard deviation.
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Table 4 were calculated as those reported in Table 3. The linearity of the ln (intensity) versus time plots were close in unity. Table 4 shows that the phosphorescence emission of saliva (non-spiked samples) has longer lifetime components than the phosphorescence emission of cocaine in saliva (spiked samples). The complex composition of saliva [1] and the lack of control in the diet of the volunteers do not allow us to attribute the phosphorescence emission to a particular component of the ¯uid. Our studies, however, show the feasibility of distinguishing the presence of cocaine in saliva by lifetime measurements. By comparing Tables 3 and 4, one can notice that the lifetime components of cocaine from saliva samples are longer than those obtained from the standard solutions. Even though saliva was dried before the RTP measurements, which caused the evaporation of water from the solid substrate, ¯uid constituents such as NaCl and protein related species [1] probably remained on the solid substrate. The phosphorescence lifetime of cocaine in saliva, therefore, was measured in the presence of other species than cocaine which may account for the observed difference. 4. Conclusion Our studies demonstrate the feasibility of using SSRTP for the detection of cocaine and benzoylecgonine in saliva. Their detection is performed on ®lter paper, which is a suitable substrate for most saliva collection methods [1]. The extraction of the parental drug and its metabolite from the solid substrate is not necessary, since direct detection of both compounds is accomplished on the paper substrate. Complete analysis can be performed with 1 ml of saliva, which is an attractive feature for cases of limited sample availability. The limits of detection estimated by the SS-RTP method were at the sub-ng level, showing the required sensitivity to reach the cut-off levels (300 ng/ml) stipulated by the National Institute on Drug Abuse (NIDA) [27]. If required, the limits of detection can be improved by optimizing NaOH, NaI and SDS concentrations for maximum phosphorescence emission. Optimization procedures for these parameters are well established in the literature, [28±32] and their application to cocaine and benzoylecgonine's analysis should be straightforward. Another possibility to lower the
levels of detection is to employ laser-excitation techniques, [33,34] which will certainly result in the detection of pg (10ÿ12 g) of analyte on the solid substrate. Finally, human saliva samples collected from nonusers with diverse diets did not show interference from concomitants in the sample. The phosphorescence spectra of cocaine and its metabolite in spiked samples were clearly differentiated from the background emission of the non-spiked samples. Although future studies should include a thorough investigation of potential interferents, the forbidden nature of the singlet-triplet electronic transition [13,14] should result into a high degree of speci®city. If existent, the interference effect of concomitants in the sample can be minimized by taking advantage of timeresolved RTP, [35] selective-external heavy-atom perturbation (SEHAP), [36] and synchronous-scanning RTP [37]. Acknowledgements This work was sponsored by the Of®ce of Health and Environmental Research, US Department of Energy, under Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corporation. References [1] D Malamud, L. Tabak, Saliva as a diagnostic fluid, Annals of the New York Academy of Sciences 694 (1993) 1±348. [2] O.R. Idowu, B. Caddy, JFSS 22 (1982) 123±135. [3] A. Warner, Clin. Chem. 35 (1989) 648±651. [4] W. Schramm, R.H. Smith, P.A. Craig, D.A. Kidwell, J. Anal. Toxicol. 16 (1992) 1±9. [5] H.W. Peel, B.J. Perrigo, N.Z. Mikhael, J. Forensic Sci. 29 (1984) 185±189. [6] G.A. Starmer, J.H. Vine, T.R. Watson, Int. J. Psychopharm. 3 (1988) 35±53. [7] L.K. Thompson, K.K. Yousefnejad, K. Kumor, M. Sherer, E.J. Cone, Anal. Toxicol. 11 (1987) 36±38. [8] K. Kato, M. Hillsgrove, D.A. Weinhold, A. Groelick, W.D. Darwin, E.J.J. Craig, Anal. Toxicol. 17 (1993) 338±341. [9] P.A. Schramm, P.A. Craig, R.H. Schmidth, G.E. Berger, Clin. Chem. 39 (1993) 481±487. [10] A.J. Jenkins, J.M. Oyler, J.E.J. Cone, Anal. Toxicol. 13 (1995) 359±374. [11] E.J. Cone, S.L. Menchen, J. Mitchell, Forensic Sci. Int. 37 (1988) 265±275.
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355 [12] E.J. Cone, W.W. Weddington, J. Anal. Toxicol. 13 (1988) 65± 68. [13] T. Vo-Dinh, Room Temperature Phosphorescence for Chemical Analysis, Wiley, New York, 1984. [14] R.J. Hurtubise, Phosphorimetry: Theory, Instrumentation and Applications, VCH, New York, 1990. [15] J.P. Alarie, T. Vo-Dinh, G. Miller, M.N. Ericson, S.R. Maddox, W. Watts, D. Eastwood, R. Lidberg, M. Dominguez, Rev. Sci. Instrum. 64 (1993) 2541±2546. [16] A.D. Campiglia, T. Vo-Dinh, Talanta 43 (1996) 1805±1814. [17] A.D. Campiglia, C.G. de Lima, Anal. Chem. 59 (1987) 2822± 2827. [18] T. Vo-Dinh, E. Lue-Yen, J.D. Winefordner, Anal. Chem. 48 (1975) 1186±1188. [19] J. McMurry, Organic Chemistry, ITP, New York, 1996. [20] J.D. Winefordner, M. Tin, Anal. Chim. Acta 32 (1965) 64±72. [21] K.F. Harbaugh, C.M. O'Donnell, J.D. Winefordner, Anal. Chem. 46 (1974) 1206±1209. [22] V.I. Stenberg, S.P. Singh, N.K. Narain, Spectrosc. Lett. 8 (1977) 639±644. [23] S.P. Singh, D. Kaufman, V.I. Stenberg, J. Heterocyclic Chem. 16 (1979) 625±631. [24] C.G. de Lima, M.M. Andino, J.D. Winefordner, Anal. Chem. 58 (1986) 2867±2869. [25] J.N. Demas, Excited State Lifetime Measurements, Academic Press, New York, 1983.
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[26] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood, Chichester, UK, 1984. [27] National Institute on Drug Abuse, Mandatory Guidelines for Federal Workplace Drug Testing Programs, Federal Register 52, 1988, 11980±11986. [28] R.Q. Aucelio, A.D. Campiglia, Mikrochim. Acta 117 (1994) 75±85. [29] R.Q. Aucelio, A.D. Campiglia, Anal. Chim. Acta 309 (1995) 345±353. [30] J. de R. Firtadp Jr, A.D. Campiglia, Michrochem. J. 52 (1995) 101±112. [31] J. de R. Furtado Jr., A.D. Campiglia, Talanta 42 (1995) 1505± 1521. [32] A.F. Arruda, A.D. Campiglia, Analyst 122 (1997) 559± 562. [33] A.D. Campiglia, D.M. Hueber, T. Vo-Dinh, Appl. Spectrosc. 50 (1996) 252±256. [34] A.D. Campiglia, J.P. Alarie, T. Vo-Dinh, Anal. Chem. 68 (1996) 1599±1604. [35] C.G. Barnes, J.D. Winefordner, Appl. Spectrosc. 38 (1984) 214±228. [36] J.R. Hooyman, T. Vo-Dinh, Anal. Chem. 51 (1979) 1915± 1921. [37] T. Vo-Dinh, R.B. Gammage, Anal. Chem. 50 (1978) 2054± 2058.
Rapid screening method for cocaine and benzoylecgonine in saliva samples A.D. Campigliaa, T. Vo-Dinhb,* a
Department of Chemistry, North Dakota State University, Fargo, ND 58105-5516, USA Life Sciences Division, Advanced Monitoring Development Group, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101, USA
b
Received 12 January 1998; received in revised form 13 April 1998; accepted 28 April 1998
Abstract We are presenting a new method for cocaine and benzoylecgonine analysis in saliva samples based on room temperature phosphorimetry. The determination of the drugs is performed on ®lter paper, which is a suitable substrate for most saliva collection procedures. Extraction from the solid substrate is not necessary, since both compounds are detected on the paper substrate. Complete analysis can be performed with only a few microliters of saliva, which is an attractive feature in cases of limited sample availability. The limits of detection are estimated at the sub-ng level, which shows the feasibility of detecting cocaine and benzoylecgonine at the cut-off levels stipulated by the National Institute on Drug Abuse (300 ng/ml). When compared to the existing methodology, the phosphorimetric method presents several advantages which include simplicity, short analysis time, low cost, and the viability of interfacing it with portable instrumentation for ®eld screening applications. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Phosphorescence; Cocaine; Benzoylecgonine; Drug diagnostics; Saliva; Drug screening
1. Introduction For the past two decades, there has been an increasing interest in the use of saliva as a matrix of analysis for therapeutic drug monitoring in a variety of clinical situations [1]. In the speci®c case of drugs of abuse monitoring, the advantages of saliva over traditional ¯uids such as urine and blood include the elimination of privacy, and to a large degree, adulteration over sample collection. The collection of saliva is a noninvasive procedure which does not require special *Corresponding author. Tel.: 00 1 615 574 6249; fax: 00 1 615 576 7651; e-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00317-1
facilities and close supervision of private functions of the subject. Although the possibility of tampering with a sample by the donor can never be completely excluded, it is unlikely that any of the commonly used methods of adulteration of urine samples can be easily applied to saliva [2±4]. Particular interest, therefore, has been expressed by law enforcement agencies for illicit drug monitoring operations and for road-side testing of potentially intoxicated drivers [2,5,6]. Recent investigations have proved various degrees of correlation among the concentration levels of cocaine and benzoylecgonine in saliva with those encountered in blood and urine samples after oral and intravenous administration [7±10]. Although
350
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355
further studies are needed to completely understand the role of saliva as a diagnostic ¯uid for drugs of abuse, [4] the positive identi®cation of cocaine and benzoylecgonine at the ng/ml level have demonstrated the potential of saliva as a non-invasive matrix of analysis. Several methods have been reported for the identi®cation and quantitation of cocaine and its metabolites in saliva. These include immunoassay, [10±12] gas chromatography, [7] and gas chromatography±mass spectrometry (GC±MS) [9,10] Currently, immunoassay is the method most frequently employed for the identi®cation of cocaine and its metabolites in biological ¯uids. Although it is a useful approach to estimate cocaine concentrations in saliva samples, it cannot be considered as a reliable quantitative assay [11]. In addition, immunoassay procedures are elaborated and time-consuming, which are important limitations for routine, in situ analysis in ®eld monitoring operations. The increasing credibility of saliva as a non-invasive matrix of analysis, associated to the need for reliable techniques in clinical and forensic science, encouraged us to develop a new screening method for cocaine and benzoylecgonine in saliva. The new method is based on solid-surface room-temperature phosphorimetry (SS-RTP), an established analytical technique which provides selective, sensitive, and simple detection of a wide variety of organic compounds [13,14]. When compared to classical methods such as immunoassay and GC-MS, the new technology offers several advantages which include simplicity and short analysis time, low cost, and the possibility of interfacing it with portable instrumentation for ®eld screening applications [15,16]. 2. Experimental 2.1. Chemicals Whatman no. 1 chromatography paper and distilled, deionized water were used throughout this work. All the chemicals were analytical-reagent grade and used without further puri®cation. Cocaine hydrochloride and benzoylecgonine standards were purchased from Sigma (USA) in methanol at 1.0 mg/ml. Benzoic acid was acquired from Aldrich and methanol from Baker,
(USA). Sodium hydroxide was obtained from EM Science (USA). Sodium dodecyl sulfate (SDS; specially puri®ed for biochemical work), lead(II) acetate, thallium(I) nitrate, cadmium(II) chloride, and sodium iodide were from Sigma (USA). 2.2. Working solutions Cocaine, benzoylecgonine, and benzoic acid solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). 2% SDS, 1 M NaI, 0.5 M Pb(CH3CH2O2), 0.1 M TlNO3, 0.5 M AgNO3, and 1.5 M CdCl2 were prepared in water. Note: use extreme caution when handling thallium salts, which are known to be extremely toxic. Saliva samples were collected from four non-user volunteers using the spitting method [1]. A portion of each sample was either spiked with cocaine or benzoylecgonine. NaOH was added to the spiked and non-spiked samples to obtain ®nal solutions with 1 M NaOH concentration. In all cases, the solvent was methanol/water 50:50 (v/v), the volume of saliva was 1 ml, and the volume of analyte solution (3.94 10ÿ4 M) was 0.1 ml. 2.3. Background reduction treatment The treatment for paper background reduction consisted in a combination of water extraction and uv irradiation [17]. Paper strips were water extracted in a soxhlet apparatus for 8 h, and exposed to uv irradiation in a photochemical reactor for the same period of time. Once treated, the solid supports were then stored in a desiccator for use in all future analyzes. 2.4. Photochemical reactor The UV irradiation of paper substrates was performed in a Rayonet photochemical reactor (Southern NE Ultraviolet, Middletown, CT, USA) using ®ve lamps with maximum wavelength of emission (em) at 254 nm and seven with em at 300 nm. 2.5. Spectrofluorimeter The phosphorescence spectra and lifetimes were determined with a Perkin-Elmer LS-50B luminescence spectrometer. Excitation and emission slit
A.D. Campiglia, T. Vo-Dinh / Analytica Chimica Acta 372 (1998) 349±355
widths were adjusted to provide 15 and 20 nm spectral resolution, respectively. In the acquisition of phosphorescence intensities, the gate time and the delay time were set at 9 and 0.3 m s, respectively. 2.6. Sample procedure Detailed procedures for the RTP assay have been discussed previously [13,14]. Although only the salient features are presented here, RTP assays usually involve simple and rapid experimental steps. All solutions were spotted on solid substrates using 2 ml micropipettes (Rainin, Model P2). In the cases were phosphorescence enhancers were used, the heavy atom and/or surfactant solutions were spotted prior to analyte deposition. In all cases, a volume of 1 ml was employed. The solid substrates were dried under an infrared lamp for 2 min. After drying, the samples were placed into a desiccator containing CaSO4 chips. All RTP measurements were performed under a ¯ow of dry nitrogen to avoid possible quenching effects from oxygen and moisture. A silica gel bed was used to remove water vapor traces from the drying ¯ow. 3. Results 3.1. Enhancement of phosphorescence emission on chromatography paper The delivery of methanolic solutions of cocaine hydrochloride and benzoylecgonine on paper substrates resulted in no RTP signal. Phosphorescence emission was induced in basic medium [18]. The analyte solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). Both compounds showed RTP emission with maximum excitation and emission wavelengths at 253 and 410 nm, respectively. Although the analyte signal intensities were relatively low (5.9410ÿ4 M analyte solutions gave signal-tobackground ratios ± IAB/IB ± approximately equal to 2), we were able to differentiate their spectral characteristics from the background signal of the paper substrate. Knowing that the basic hydrolysis of cocaine and benzoylecgonine leads to the formation of benzoic acid and ecgonine, [19] and considering that phos-
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phorescence emission at room temperature is observed from saturated organic molecules with a certain degree of conjugation, we investigated the RTP characteristics of benzoic acid in 1 M NaOH (methanol/ water 50:50 v/v). As expected, its phosphorescence spectrum was equivalent to the one observed for cocaine hydrochloride and benzoylecgonine in basic medium. Although these two compounds have the benzoate chromophore in their molecular structure, their interaction with the solid substrate might not provide the analyte rigidity required for strong phosphorescence emission at room temperature. At 77 K, however, the radiationless deactivation of cocaine's triplet state is minimized by the frozen matrix, and the phosphorescence emission from the benzoate group is readily observed [20±23]. 3.2. External heavy-atom effect Five inorganic salts (1 M NaI, 0.5 M Pb(CH3CH2O2)2, 0.1 M TlNO3, 0.5 M AgNO3, and 1.5 M CdCl2) were tested as phosphorescence enhancers [13,14]. Table 1 shows the results obtained for cocaine hydrochloride in basic medium. Cd(II) and Ag(I) ions did not induce RTP emission from the studied compounds (IAB/IB1). Tl (I) promoted RTP enhancement (IAB/IB>1), but its enhancing effect was lower than the one observed with iodide ions. As expected, similar results were obtained for benzoylecgonine. All further studies, therefore, were Table 1 Heavy-atom effect on the RTP characteristics of cocaine hydrochloride in basic mediuma Inorganic saltb
excc (nm)
emd (nm)
IAB/IBe
1 M NaI 1.5 M CdCl2 0.1 M TlNO3 0.5 M AgNO3
253 Ð 230 Ð
410 Ð 400 Ð
7.80.9 1.00.1 3.80.8 1.00.2
a
2.9710ÿ4 M solution of cocaine hydrochloride in 1 M NaOH (methanol/water 50:50 v/v) was employed. One ml sample volume was spotted on low background chromatography paper. b All heavy-atom solutions were prepared in methanol/water (30:70 v/v). One ml heavy-atom solution was employed. c Maximum excitation wavelength. d Maximum emission wavelength. e IABanalyte signal; IBbackground signal. Six measurements of analyte signals and their respective blanks were performed to obtain the analyte-to-background signal ratios.
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performed employing 1 M NaI as a phosphorescence enhancer. 3.3. Surfactant effect As a tentative means of improving iodide ef®ciency, we tested SDS in the matrix of the phosphor [24]. A 2% SDS solution was spotted on the chromatography paper prior to 1 M NaI and analyte deposition. A signi®cant increase in the IAB/IB ratio was observed in the presence of surfactant. For a 2.9710ÿ4 M cocaine hydrochloride solution (prepared in 1 M NaOH; methanol/ water 50:50 v/v), an IAB/IB 15.71.6 was obtained (NABNB9). This ratio is approximately two times higher than the one obtained in the absence of surfactant (see Table 1), showing that SDS increases the phosphorescence enhancing ef®ciency of iodide ions. 3.4. Analytical figures of merit Table 2 shows the analytical ®gures of merit of cocaine hydrochloride and benzoylecgonine under experimental conditions for maximum phosphorescence emission. Each phosphorescence intensity plotted on the calibration graph was the average of six data points. The linear dynamic ranges (LDR) extended over three orders of magnitude. The slopes Table 2 Analytical figures of merit of cocaine hydrochloride and benzoylecgonine obtained under experimental conditions for maximum phosphorescence emissiona Compoundb
LDRc (ng)
Slope log±log
Cocaine hydrochloride 0.16±50 1.05 Benzoylecgonine 0.22±50 0.94 a
Correlation ALODd coefficient (ng) 0.9944 0.9987
0.16 0.22
Maximum phosphorescence emission was obtained on solid substrates successively spotted with 1 ml of 1 M NaI and 1 ml of a 2% aqueous solution of sodium dodecyl sulfate. The measurement wavelengths were 253 and 410 nm. b The analyte solutions were prepared in 1 M NaOH (methanol/ water 50:50 v/v). c LDRlinear dynamic range. It was estimated by dividing the upper linear concentration by the limit of detection. d ALODabsolute limit of detection. Sample volume: 1 ml. The LOD values were estimated from the equation CLksb/m, where the standard deviation (sb) from 16 blank determinations, the slope of the calibration curve (m) and k 3 were employed.
Table 3 Phosphorescence lifetimes of cocaine hydrochloride, benzoylecgonine, and benzoic acid on chromatography paper treated for background reductiona Compoundb
exc/em (nm)
p; Short componentc (m s)
p; Long componentd (m s)
Cocaine hydrochloride Benzoylecgonine Benzoic acid
253/410 253/410 253/410
0.600.05 0.620.01 0.680.05
2.260.20 2.140.48 2.100.10
a Solid substrates spotted with 1 ml of 1 M NaI and 1 ml of 2% sodium dodecyl sulfate were employed. b All solutions were prepared in 1 M NaOH (methanol/water 50:50 v/v). c Delay times were from 0.1 to 1.1 m s. Gate time was 0.1 m s. d Delay times were from 2 to 9 m s. Gate time was 0.1 m s.
of the log±log plots were close to one, which showed a linear relationship between phosphorescence intensity and analyte concentration. The correlation coef®cients of the calibration curves were close in unity, indicating a satisfactory precision of measurement. For both compounds, the relative standard deviations were better at medium and higher concentrations, varying from 4.8% (for a 10 mg/ml cocaine solution) to 8.7% (for a 50 mg/ml benzoylecgonine solution). The phosphorescence lifetimes of cocaine hydrochloride, benzoylecgonine, and sodium benzoate in basic medium are shown in Table 3. The exponential decays were complex and included short and long decaying components. Their values were calculated by the stripping method suggested by Demas [25]. The least-squares analysis of the long decay components yield correlation coef®cients close in unity (higher than 0.99). The lifetime values in Table 3 are based on six determinations (N6) of the analyte decay and the blank decay. For a con®dence interval of 95% (P0.05), the statistical equivalence [26] among the lifetimes of the three compounds support our initial assumption that sodium benzoate is the phosphorescent species in basic medium. 3.5. Determination of cocaine hydrochloride and benzoylecgonine in saliva Saliva samples were collected from four non-user volunteers. Two tobacco smoker volunteers were included in the test group. The RTP measurements were performed with 1 ml of saliva deposited on
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Fig. 1. (A) Excitation and (B) emission spectra of saliva samples (a) spiked and (b) non-spiked with cocaine hydrochloride. The amount of cocaine hydrochloride deposited on the paper substrate is approximately 11 ng.
chromatography paper previously treated with 1 M NaI and 2% SDS. The phosphorescence spectra of the spiked and non-spiked samples were compared for the four individuals. Fig. 1 shows the RTP spectra of cocaine in saliva. The presence of cocaine in the spiked sample was clearly detected by the phosphorescence bands with maximum excitation and emission wavelengths at 258/413 nm, respectively (see Fig. 1, curve A). The phosphorescence spectrum of saliva showed a broad excitation band with plateau extended from 240 to 280 nm, and a maximum of emission at
440 nm (Fig. 1, curve B). The contribution of saliva to the background signal of the solid substrate was approximately 15%. This contribution did not deteriorate cocaine's limit of detection in saliva, which was estimated at the ng level. Equivalent results were obtained for benzoylecgonine (P0.05; N6) [26]. No signi®cant differences were observed in the spectral characteristics of the saliva samples collected from the four volunteers. Table 4 summaries the lifetimes of cocaine phosphorescence emission in saliva samples. The values in
Table 4 Phosphorescence lifetime measurements of cocaine in saliva samplesa Volunteer
1 2 3 Average RSD (%)b a b
Spiked samples
Non spiked samples
Short component (ms)
Long component (ms)
Short component (ms)
Long component (ms)
0.83 1.00 0.90 0.910.08 9.3
4.17 4.22 4.25 4.210.04 1.0
2.38 2.81 3.06 2.750.34 12.4
5.72 5.62 5.70 5.680.05 0.9
For experimental conditions and instrumental parameters see Table 2. RSDrelative standard deviation.
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Table 4 were calculated as those reported in Table 3. The linearity of the ln (intensity) versus time plots were close in unity. Table 4 shows that the phosphorescence emission of saliva (non-spiked samples) has longer lifetime components than the phosphorescence emission of cocaine in saliva (spiked samples). The complex composition of saliva [1] and the lack of control in the diet of the volunteers do not allow us to attribute the phosphorescence emission to a particular component of the ¯uid. Our studies, however, show the feasibility of distinguishing the presence of cocaine in saliva by lifetime measurements. By comparing Tables 3 and 4, one can notice that the lifetime components of cocaine from saliva samples are longer than those obtained from the standard solutions. Even though saliva was dried before the RTP measurements, which caused the evaporation of water from the solid substrate, ¯uid constituents such as NaCl and protein related species [1] probably remained on the solid substrate. The phosphorescence lifetime of cocaine in saliva, therefore, was measured in the presence of other species than cocaine which may account for the observed difference. 4. Conclusion Our studies demonstrate the feasibility of using SSRTP for the detection of cocaine and benzoylecgonine in saliva. Their detection is performed on ®lter paper, which is a suitable substrate for most saliva collection methods [1]. The extraction of the parental drug and its metabolite from the solid substrate is not necessary, since direct detection of both compounds is accomplished on the paper substrate. Complete analysis can be performed with 1 ml of saliva, which is an attractive feature for cases of limited sample availability. The limits of detection estimated by the SS-RTP method were at the sub-ng level, showing the required sensitivity to reach the cut-off levels (300 ng/ml) stipulated by the National Institute on Drug Abuse (NIDA) [27]. If required, the limits of detection can be improved by optimizing NaOH, NaI and SDS concentrations for maximum phosphorescence emission. Optimization procedures for these parameters are well established in the literature, [28±32] and their application to cocaine and benzoylecgonine's analysis should be straightforward. Another possibility to lower the
levels of detection is to employ laser-excitation techniques, [33,34] which will certainly result in the detection of pg (10ÿ12 g) of analyte on the solid substrate. Finally, human saliva samples collected from nonusers with diverse diets did not show interference from concomitants in the sample. The phosphorescence spectra of cocaine and its metabolite in spiked samples were clearly differentiated from the background emission of the non-spiked samples. Although future studies should include a thorough investigation of potential interferents, the forbidden nature of the singlet-triplet electronic transition [13,14] should result into a high degree of speci®city. If existent, the interference effect of concomitants in the sample can be minimized by taking advantage of timeresolved RTP, [35] selective-external heavy-atom perturbation (SEHAP), [36] and synchronous-scanning RTP [37]. Acknowledgements This work was sponsored by the Of®ce of Health and Environmental Research, US Department of Energy, under Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corporation. References [1] D Malamud, L. Tabak, Saliva as a diagnostic fluid, Annals of the New York Academy of Sciences 694 (1993) 1±348. [2] O.R. Idowu, B. Caddy, JFSS 22 (1982) 123±135. [3] A. Warner, Clin. Chem. 35 (1989) 648±651. [4] W. Schramm, R.H. Smith, P.A. Craig, D.A. Kidwell, J. Anal. Toxicol. 16 (1992) 1±9. [5] H.W. Peel, B.J. Perrigo, N.Z. Mikhael, J. Forensic Sci. 29 (1984) 185±189. [6] G.A. Starmer, J.H. Vine, T.R. Watson, Int. J. Psychopharm. 3 (1988) 35±53. [7] L.K. Thompson, K.K. Yousefnejad, K. Kumor, M. Sherer, E.J. Cone, Anal. Toxicol. 11 (1987) 36±38. [8] K. Kato, M. Hillsgrove, D.A. Weinhold, A. Groelick, W.D. Darwin, E.J.J. Craig, Anal. Toxicol. 17 (1993) 338±341. [9] P.A. Schramm, P.A. Craig, R.H. Schmidth, G.E. Berger, Clin. Chem. 39 (1993) 481±487. [10] A.J. Jenkins, J.M. Oyler, J.E.J. Cone, Anal. Toxicol. 13 (1995) 359±374. [11] E.J. Cone, S.L. Menchen, J. Mitchell, Forensic Sci. Int. 37 (1988) 265±275.
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